A surface loop (25/50-kDa loop) near the nucleotide pocket of myosin has been proposed to be an important element in determining the rate of ADP release from myosin, and as a consequence, the rate of actin-myosin filament sliding (Spudich, J. A. (1991) Nature 372, 515-518). To test this hypothesis, loops derived from different myosin II isoforms that display a range of actin filament sliding velocities were inserted into a smooth muscle myosin backbone. Chimeric myosins were produced by baculovirus/Sf9 cell expression. Although the nature of this loop affected the rate of ADP release (up to 9-fold), in vitro motility (2.7-fold), and the V max of actin-activated ATPase activity (up to 2-fold), the properties of each chimera did not correlate with the relative speed of the myosin from which the loop was derived. Rather, the rate of ADP release was a function of loop size/flexibility with the larger loops giving faster rates of ADP release. The rate of actin filament translocation was altered by the rate of ADP release, but was not solely determined by it. Through a combination of solute quenching and transient fluorescence measurements, it is concluded that, as the loop gets smaller, access to the nucleotide pocket is more restricted, ATP binding becomes less favored, and ADP binding becomes more favored. In addition, the rate of ATP hydrolysis is slowed.Resolution of the atomic structure of the myosin II motor from crystallographic x-ray diffraction data has created interest in several flexible loops on its surface. These include the "HCM" loop (1, 2) and two loops that were not resolved in the crystal structures and are the sites of proteolytic cleavage: the loops at the 25/50-kDa junction and 50/20-kDa junction. These junctional loops also have been referred to as loop 1 (25/50-kDa loop) and loop 2 (50/20-kDa loop). The HCM loop is so called because a number of mutations at its base are associated with the human disease, hypertrophic cardiomyopathy (3). Furthermore, some members of the myosin family are regulated by phosphorylation of this loop (4, 5). Loops 1 and 2 have been proposed to be major determinants of the kinetic properties of myosin (6). Both loops are highly variable in sequence and length among members of the myosin II family. If this variability underlies a large degree of the kinetic diversity among myosins, then it would reveal an evolutionary strategy for kinetic tuning that would involve regions of the molecule outside of the core that might not have interactions with the backbone of the myosin motor. This could allow a rapid evolutionary divergence of motor properties without altering the core structure or the motor's basic function.The suggestion of a fundamental role for the junctional loops of myosin was based on a study of loop 2, which is located at the actin interface (7) and on the demonstration of functional differences for naturally occurring isoforms involving alternations in loop 1 (25/50-kDa loop) (8). The former study involved the creation of chimeric myosin II molecules that we...

Myosin subfragment 1 (S-1) can be fractionated into two isozymes, (A1)S-1 containing alkali light chain 1 and (A2)S-1 containing alkali light chain 2. The predominant difference in the behavior of the two isozymes of S-1 is that, at low ionic strength, the actin concentration required for half-maximal ATPase activity is considerably lower for (A1)S-1 than for (A2)S-1; that is, the apparent binding constant KATPase for (A1)S-1 is greater than KATPase for (A2)S-1 [Weeds, A.G., & Taylor, R.S. (1975) Nature (London) 257, 54-56]. This difference disappears at high ionic strength [Wagner, P. D., Slater, C. S., Pope, B., & Weeds, A.G. (1979) Eur. J. Biochem. 99, 385-394]. In the present study we investigated whether the difference in the KATPase values of (A1)S-1 and (A2)S-1 is due to a difference in the actual affinity of these S-1 isozymes for actin. Binding was measured in the presence of ATP and AMP-PNP and in the absence of nucleotide at varied ionic strengths. We found that at low ionic strength where KATPase is several times stronger for (A1)S-1 than for (A2)S-1, the binding of (A1)S-1 to actin is correspondingly stronger than that of (A2)S-1 irrespective of the nucleotide present. Furthermore, as the ionic strength is increased, just as the difference between the KATPase values for (A1)S-1 and (A2)S-1 disappears so too does the difference in the affinity of the two isozymes for actin.(ABSTRACT TRUNCATED AT 250 WORDS)

The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP contributes to protein folding homeostasis by engaging unfolded client proteins in a process that is tightly coupled to ATP binding and hydrolysis. The inverse correlation between BiP AMPylation and the burden of unfolded ER proteins suggests a post-translational mechanism for adjusting BiP's activity to changing levels of ER stress, but the underlying molecular details are unexplored. We present biochemical and crystallographic studies indicating that irrespective of the identity of the bound nucleotide AMPylation biases BiP towards a conformation normally attained by the ATP-bound chaperone. AMPylation does not affect the interaction between BiP and J-protein co-factors but appears to allosterically impair J protein-stimulated ATP-hydrolysis, resulting in the inability of modified BiP to attain high affinity for its substrates. These findings suggest a molecular mechanism by which AMPylation serves as a switch to inactivate BiP, limiting its interactions with substrates whilst conserving ATP.

The Lymn-Taylor model for the actomyosin ATPase suggests that during each cycle of ATP hydrolysis the complex of myosin subfragment 1 (S-i) with actin must dissociate into S-1ATP plus actin before ATP hydrolysis can occur. In the present study we tested whether such a mandatory detachment step occurs by measuring the effect of actin on the rate and magnitude of the ATP hydrolysis step (initial Pi burst) and on the steady-state AT- only when M-ATP is detached from actin. This feature of the model requires myosin to detach from actin during each cycle of ATP hydrolysis, a point that has major implications for the mechanism of cross-bridge action in vivo (2). Therefore, it is important to determine whether ATP hydrolysis is indeed prevented when M-ATP is bound to actin, particularly because recent evidence suggests that a detachment step may not always be required when actomyosin hydrolyzes ATP in vitro (3, 4). The most direct method of determining whether the ATPase cycle includes a mandatory detachment step is to study the effect of actin on the ATP hydrolysis step (initial Pi burst) because in the Lymn-Taylor model this step occurs only with the S-1 detached from actin. In the present study we determined the effect of actin on the rate and magnitude of the initial Pi burst.In addition, we determined the effect of very high actin concentration on the steady-state ATPase rate. The results of all of these experiments show that, if anything, the rate of the ATP hydrolysis step is even faster when S-1 is bound to actin than when it is dissociated from actin. This indicates that S-1 is not required to detach from actin during each cycle of ATP hydrolysis. MATERIALS AND METHODSin each cl o MM .f Myosin (5), actin (6), and chymotryptic S-1 (7) were prepared burst appears to as described and their protein concentrations were determined i than when it is spectrophotometrically (3). Measurements of the rate of fluorescence enhancement and the turbidity of acto-S-1 solutions were carried out in a stoppedtic studies using flow apparatus as described (3). Quenched-flow experiments neromyosin and were carried out in a Durram D-132 multimixer, as described posed the follow-(8) except that, to facilitate mixing, the pressure was held constant at 80 psi (552 kilopascals) and the quench time was varied by changing the length of the reaction tubing. ATPase rate and the rate and magnitude of the initial Pi burst as predicted by the Lymn-Taylor model (scheme I). The derivation of these equations will be presented elsewhere.We assume that a rapid equilibrium exists between M-ATP

Although there is agreement that actomyosin can hydrolyze ATP without dissociation of the actin from myosin, there is still controversy about the nature of the rate-limiting step in the ATPase cycle. Two models, which differ in their rate-limiting step, can account for the kinetic data. In the four-state model, which has four states containing bound ATP or ADP . Pi, the rate-limiting step is ATP hydrolysis (A . M . ATP in equilibrium A . M . ADP . Pi). In the six-state model, which we previously proposed, the rate-limiting step is a conformational change which occurs before Pi release but after ATP hydrolysis. A difference between these models is that only the four-state model predicts that almost no acto-subfragment 1 (S-1) . ADP . Pi complex will be formed when ATP is mixed with acto . S-1. In the present study, we determined the amount of acto . S-1 . ADP . Pi formed when ATP is mixed with S-1 cross-linked to actin [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Nature (London) 292, 301-306]. The amount of acto . S-1 . ADP . Pi was determined both from intrinsic fluorescence enhancement and from direct measurement of Pi. We found that at mu = 0.013 M, the fluorescence magnitude in the presence of ATP of the cross-linked actin . S-1 preparation was about 50% of the value obtained with S-1, while at mu = 0.053 M the fluorescence magnitude was about 70% of that obtained with S-1.(ABSTRACT TRUNCATED AT 250 WORDS)